3-Dimensional Tissue Construct Assays for Ocular Irritation

3-D human tissue constructs are organotypic in vitro tissue models of the human corneal epithelium (HCE) used to evaluate the potential ocular irritation of a test material. Tissue viability is determined using the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The reduction of MTT in treated cultures is expressed as a percentage relative to negative control cultures.

The 3-D ocular constructs are made from human epithelial cells, which are cultured on specially designed cell culture inserts. The cells differentiate to form a multi-layered structure which closely models the corneal epithelium (see picture below).

MTT, a vital dye, is actively taken up by the tissues and subsequently reduced in the mitochondria of living cells. This chemical reaction produces a purple-colored formazan within the cells, causing the live tissues to turn deep purple in color. The colored dye can be extracted from the cells and the absorbance read spectrophotometrically. For specific assay procedures, please see Step-by-Step.

Assay Design: Quick Facts

Assay Model: organotypic 3-D tissue constructs

Endpoint: ET50 (or t50) (the exposure time that induces a 50% reduction in viability, relative to negative controls)

Each assay includes a positive and negative control. For more information about testing your materials using this assay, please see Applications. Specialized protocols may be prepared as requested through consultation with an IIVS Study Director.